1. Technical Field
The present invention relates to vaccines for equine influenza virus, and, more particularly, to a DNA vaccine comprising the HA1 encoding sequence of equine-2 influenza virus which may be administered intranasally of a lower than typical dosage to elicit good mucosal immunity.
2. Background
Equine influenza virus (EIV) is the leading etiological agent for upper respiratory infections in horses. It has been implicated as the cause of epidemic outbreaks of respiratory disease in the horse for centuries. Spread of the virus is rapid and morbidity is extremely high. Infected horses develop typical “flu” symptoms: rapid onset of respiratory distress, coughing, fever, and mucous discharge. In rare cases, fatalities result from secondary bacterial bronchial pneumonia. Although mortality rate is low, the effect of an equine influenza virus infection is significant. It is estimated that the suspension of horse racing in a 1992 Hong Kong outbreak resulted in a loss of US$120 million in revenue. The economic importance in other equine sports may be less, but outbreaks of equine influenza have interrupted international equine events on several occasions. An infected horse without clinical signs but undergoing strenuous training may suffer long term consequences such as reduced pulmonary function. Clinically ill horses suffer the obvious disadvantage of losing training time.
Equine influenza virus is type A influenza virus, a member of Orthomyxoviridae. The viral genome consists of eight segments of negative-stranded RNA. The viral capsid is enclosed in a lipid envelope anchoring two surface viral glycoproteins: hemagglutinin (HA) and neuraminidase (NA). HA is believed to be the most antigenic viral protein of EIV. HA has a molecular weight of approximately 77 kD. This viral protein is synthesized as HA0, and it is cleaved by protease action and subsequent reduction of the single disulfide bond into an amino terminal HA1 portion (50 kD) and a carboxy terminal HA2 portion (27 kD). The HA2 portion is anchored onto the lipid bilayer of a membrane, and HA1 portion is bound to HA2 by non-covalent linkages. The hemagglutinin is involved in binding of the virus to the receptor at the host cell membrane, leading to the subsequent penetration and uncoating of the virus, hence initiating a viral replication. A major goal of vaccination is to induce immunity towards this viral encoded molecule.
There are two subtypes of equine influenza viruses. Type 1, or equine-1 influenza virus (H7N7), has not been isolated in developed countries for the last 15 years. Equine-2 influenza virus (H3N8), however, continues to circulate around the world despite massive vaccination programs. The success of H3N8 virus is probably due to antigenic drift: sequential changes of the antigenicity of HA by amino acid substitution [1]. Recent isolates of equine-2 influenza viruses can be classified into either “American” lineage or “Eurasian” lineage. Furthermore, more recent equine-2 influenza virus has diverged into multiple lineages [2], and that at least in North America, two evolutionary lineages circulate in alternate year [3].
Current EIV vaccines typically consist of formalin or β-propiolactone inactivated whole viruses. The antigenic constituent is composed of equine influenza virus type 1 and type 2. A/Eq/Prague/56 is the only vaccine strain for type 1, whereas, type 2 constituents are the prototype A/Eq/Miami/63 and a later strain such as A/Eq/Kentucky/81 or A/Eq/Fontainebleau/79. A recent meeting of WHO/OIE Consultations on Control of Equine Influenza affirmed the earlier recommendations that vaccines should include both an “American” virus (A/Eq/Kentucky/94) and a “Eurasian” virus (A/Eq/Newmarket/2/93), and that the prototype A/Eq/Miami/63 should be discontinued [4].
In recent prospective study, Morley et. al. [5] have shown that current commercial vaccines do not protect against virus infection, and only have marginal effect in the suppression of clinical symptoms. The lack of protection offered by current commercial vaccines is due to one, or more, of a combination of the following factors:                lack of imununogenicity;        poor choice of vaccine strains; and/or        eliciting an inappropriate immunity.        
The continued evolution of equine-2 influenza virus (H3N8) requires periodic updating of the vaccine strain to elicit protective immunity. However, there is a wide spectrum of vaccine strain choices among different vaccine manufacturers.
Immunity generated by an earlier EIV will not be protective against later isolates due to a change of the antigenicity of HA, a result of amino acid substitutions (antigenic drift). This characteristic of the virus is the major obstacle to a “fail-proof” effective vaccine. Updating of vaccine by replacing with more recent virus strains and in a more frequent intervals had been recommended [6]. Some manufacturers still keep outdated virus strains in their products. Although antibodies specific for equine influenza virus are elicited, however, these vaccines are problematic. First, serum antibody level serves as a poor indicator for protection. Second, as the circulating virus strains are sufficiently different from the vaccine strain, there is minimal cross-reactivity. A “partial” immunity elicited by such outdated vaccine renders an infected host a non-symptomatic carrier, that is, the host is infected, but because of the partial immunity, clinical symptoms are suppressed. These infected hosts are not recognized, which facilitates the spread of the virus.
Influenza virus initiates infection by attachment to the ciliated epithelial cells at the upper respiratory tract. Therefore, mucosal antibodies provide an effective defense against the virus. In fact, the importance of nasal antibodies in protection against equine influenza virus has been recognized for many years. In a mouse model, it has been shown that transfer of IgA confers protection against influenza virus infection [7].
Whereas current vaccines elicit serum antibodies, none target mucosal immunity. The correlation between serum antibodies level and vaccine efficacy is unclear, due to the lack of standardization of the measurement for both the antigen and the antibodies [8].
Several strategies, including the use of immune stimulating complexes (ISCOMs) [9], and by direct inoculation to the mucosal area [10], have been used to boost mucosal immune response to current vaccines for equine influenza virus. However, the results showed only limited improvements using these strategies.
A recently licensed vaccine from Heska Corp. (Fort Collins, Colo.), based on recombinant cold-adapted (temperature-sensitive mutant) and attenuated equine influenza virus, is an attempt to elicit mucosal immunity. The vaccine is administered by intranasal inoculation to elicit mucosal immunity. Direct inoculation of cold-adapted attenuated virus to the mucosal site intranasally provides strong stimulation of the mucosal-associated lymphoid tissues (MALT). Therefore, this vaccine is highly immunogenic and elicits mucosal immunity. However, since the vaccine is based on recombinant virus through re-assortment, updating the vaccine requires re-engineering of the cold-adapted attenuated virus. All necessary safety and potency testing has to be done before the updated vaccine can be licensed.
The field of DNA vaccine, or genetic immunization, is a rapidly emerging technology. It was a serendipitous discovery that when a DNA plasmid containing the coding sequences of a protein is injected intramuscularly into a mouse, not only was the antigen expressed, but an immune response to the antigen was also elicited [11, 12]. It is believed that cells take up the DNA plasmid in vivo in a manner similar to that of a DNA transfection in vitro. The DNA plasmid does not replicate inside the host cells, but the encoded antigen is transcribed and translated by the host cell. The antigen is either expressed on the cell surface or secreted, and an immune response is elicited [13]. This new immunization methodolog has been shown to be effective by many investigators, and for a wide spectrum of infectious agents, including influenza virus in general [14], and specific for equine influenza virus [15]. It has been shown that a DNA vaccine expressing the HA gene of A/Eq/Kentucky/81, after administered via skin and mucosa, protected horses against a homologous virus challenge [15].
In the patent art, vaccines and methodologies against EIV are described in U.S. Pat. Nos. 6,482,414; 6,436,408; 6,398,774; 6,177,082; 6,045,790; 4,920,213; 4,693,893; 4,689,224; 4,683,137; 4,631,191; and 4,619,827, all of which are incorporated herein by reference. U.S. Pat. Nos. 4,920,213 and 4,631,191 are directed to recombinant vaccines for immunizing horses against equine influenza virus. DNA sequences encoding the HA and NA glycoproteins from two strains were used to construct vaccinia carried vaccines, to design synthetic peptides for primer and booster administration, and to permit recombinant synthesis of HA and/or NA protein based vaccines.
An ideal vaccine for equine influenza virus, by addressing the above deficiency of current vaccines, should be highly immunogenic, elicit a mucosal immunity, and be amenable to easy “updating”.